The aim of this project is to characterize the expression of the putative testis-determining gene, Sry, in preimplantation mouse embryos and to ask whether this expression is necessary for male sex determination. The level of expression of Sry mRNA will be quantified at several preimplantation stages using a polymerase chain reaction (PCR)-based procedure that was developed previously in my lab (Gaudette and Crain, 1991). To test whether Sry expression in early embryos causes male sex determination 'antisense' nucleic acids will be introduced to inactivate the Sry mRNA, and the sex of live offspring that derive from the treated embryos will be determined after transfer to host females. The appearance of XY females would indicate that functional in mRNA is required for male sex determination. Several "antisense" strategies will be tested: (1) incubation of embryos in media containing chemically modified and unmodified oligodeoxynucleotides (ODNs); (2) microinjection of modified and unmodified ODNs; (3) injection of antisense RNA synthesized from riboprobe vectors., Full length cDNA clones of Sry mRNA will be isolated from blastocyst and testis RNA, and their sequences compared to determine whether differential splicing or promotor use occurs to produce different proteins in the two tissues. The complete sequence of the cDNA clones will also be compared to the genomic DNA to determine the structure of the gene and to define the location of upstream sequences. With the long term goal of learning how the Sry gene is activated in early embryogenesis, in vivo competition assays will be used to locate the cis-regulatory elements associated with this gene.